His...Asp catalytic dyad of ribonuclease A: structure and function of the wild-type, D121N, and D121A enzymes

Abstract

The side chains of histidine and aspartate residues form a hydrogen bond in the active sites of many enzymes. In serine proteases, the His...Asp hydrogen bond of the catalytic triad is known to contribute greatly to catalysis, perhaps via the formation of a low-barrier hydrogen bond. In bovine pancreatic ribonuclease A (RNase A), the His...Asp dyad is composed of His119 and Asp121. Previously, site-directed mutagenesis was used to show that His119 has a fundamental role, to act as an acid during catalysis of RNA cleavage [Thompson, J. E., and Raines, R. T. (1994) J. Am. Chem. Soc. 116, 5467-5468]. Here, Asp121 was replaced with an asparagine or alanine residue. The crystalline structures of the two variants were determined by X-ray diffraction analysis to a resolution of 1.6 A with an R-factor of 0.18. Replacing Asp121 with an asparagine or alanine residue does not perturb the overall conformation of the enzyme. In the structure of D121N RNase A, Ndelta rather than Odelta of Asn121 faces His119. This alignment in the crystalline state is unlikely to exist in solution because catalysis by the D121N variant is not compromised severely. The steady-state kinetic parameters for catalysis by the wild-type and variant enzymes were determined for the cleavage of uridylyl(3'-->5')adenosine and poly(cytidylic acid), and for the hydrolysis of uridine 2',3'-cyclic phosphate. Replacing Asp121 decreases the values of kcat/Km and kcat for cleavage by 10-fold (D121N) and 10(2)-fold (D121A). Replacing Asp121 also decreases the values of kcat/Km and kcat for hydrolysis by 10(0. 5)-fold (D121N) and 10-fold (D121A) but has no other effect on the pH-rate profiles for hydrolysis. There is no evidence for the formation of a low-barrier hydrogen bond between His119 and either an aspartate or an asparagine residue at position 121. Apparently, the major role of Asp121 is to orient the proper tautomer of His119 for catalysis. Thus, the mere presence of a His...Asp dyad in an enzymic active site is not a mandate for its being crucial in effecting catalysis.

Figure 1

Putative mechanism for the transphosphorylation (A) and hydrolysis (B) reactions catalyzed by RNase A (33). In the hydrolysis reaction, the hydrogen bonds between Asp121⋯His119⋯H₂O resemble those in the renown catalytic triad of serine proteases.

Figure 2

Structure of the active site of crystalline D121N RNase A (A) and D121A RNase A (B). Structures were determined by X-ray diffraction analysis to a resolution of 1.6 Å. Hydrogen bonds of ≤3.2 Å are labeled. Enzymes were crystallized at pH 5.2 (D121N RNase A) or pH 5.3 (D121A RNase A).

Figure 3

Structural differences between D121N RNase A and D121A RNase A and their semisynthetic analogs. (A) Stereoview of the overlap of the active sites of D121N RNase A (thick red lines) and the semisynthetic D121N analog [thin blue lines; (30)]. The sD121N analog contains a sulfate ion in its active site. (B) Stereoview of the overlap of the active sites of D121A RNase A (thick red lines) and the sD121A analog [thin blue lines; (30)]. D121A RNase A contains an acetate ion in its active site, and the sD121A analog contains a sulfate ion in its active site. Water molecules are depicted for the variants (red closed spheres) and semisynthetic analogs (blue open spheres). This figure was created with the program MOLSCRIPT (93).

Figure 4

Plot of log(k_cat/K_m) vs pH for the hydrolysis of cUMP by wild-type RNase A (E), D121N RNase A (I), and D121A RNase A (D). The curves are nonlinear least-squares fits of the data to eq 1. To simplify comparisons, the values of k_cat/K_m for D121N RNase A and D121A RNase A were multiplied by scaling factors of 2.2. and 13, respectively.

Figure 5

Plot of log(k_cat) vs pH for the hydrolysis of cUMP by wild-type RNase A (E), D121N RNase A (I), and D121A RNase A (D). The curves are nonlinear least-squares fits of the data to eq 2. To simplify comparisons, the values of k_cat for D121N RNase A and D121A RNase A were multiplied by scaling factors of 1.6 and 16, respectively.

Figure 6

Plot of –log(K_p) vs pH for the hydrolysis of cUMP by wild-type RNase A (E), D121N RNase A (I), and D121A RNase A (D). The curves are nonlinear least-squares fits of the data to eq 3. No scaling factors were applied to these data.